Under normal conditions, nitric oxide (NO) is a short-lived, unstable gaseous substance. Its instability is due to the unpaired electron of nitrogen. As an unstable substance with an unpaired electron, nitric oxide can be described as a free radical. However, compared with typical free radicals (e.g. hydroxyl radical or superoxide), whose life-time is in the order of milliseconds, nitric oxide is relatively stable. Typically, it is converted to a more stable chemical species within seconds of its production. Thus, for example, if gaseous nitric oxide contacts air, it reacts rapidly with oxygen to generate nitrogen dioxide as follows:2NO+O2→2NO2→N2O4 
Under some conditions, for instance in pure gaseous state, NO can be stored without significant losses for a very long time. NO is a very hydrophobic compound and its solubility in water is therefore limited. Maximum solubility in water achievable under normal conditions is approximately 1.7 mM, the solubility being similar to that of oxygen. The oxidation of dissolved nitric oxide by dissolved oxygen occurs in aqueous solutions. Nevertheless, given the rate constants and low concentrations of dissolved NO and O2 this reaction is considerably less rapid than in the gaseous state, where the concentration of oxygen is very high.
Nitric oxide can be produced by chemical reduction of nitrous acid. Many different reducing agents can be used to reduce nitrous acid, physiologically acceptable examples of such reducing agents include iodide anion, ascorbic acid, butylated hydroquinone, tocopherol etc. Nitrous acid is a weak acid with pKa 3.4. This means that at pH 3.4, nitrous acid exists as an equimolar mixture of nitrous acid (HNO2) and nitrite (NO2−). At higher pH, the equilibrium shifts in favour of nitrite anion; at lower pH the equilibrium shifts in favour of nitrous acid. Since only nitrous acid can be chemically reduced to nitric oxide the efficiency of converting nitrite into nitric oxide increases with decreasing pH. So, whilst at pH 6 the rate of such conversion is negligible, it proceeds slowly at pH 5 and is very rapid at pH<4 and especially at pH<3.
A special category of reducing agents that react with nitrite in an acidic environment are thiols. Reaction between thiols and nitrite in an acidic environment does not result in nitrous acid reduction and immediate generation of nitric oxide, as in the case of other reducing agents. Instead, thiols are nitrosylated by nitrosonium cation (NO+) which is another species generated from nitrite in acidic conditions.
Nitric oxide has a multitude of effects in living tissues. The mechanism of these effects is nearly always based on interaction of nitric oxide either with a metal component (typically iron) or with thiol groups of key enzymes and other proteins. Depending on the particular enzyme, such interaction can lead to either activation or inhibition of the protein. An example of an effect based on the activation of an enzyme is that of vasodilatation: nitric oxide binds to the haem iron of the enzyme guanylate cyclase, which results in conformational change exposing the catalytic site of the enzyme. This leads to catalytic conversion of GTP to cGMP. This conversion initiates the whole cascade of reactions leading to protein phosphorylation and muscle relaxation (vasodilatation). Other effects based on activation of enzymes or growth factors by nitric oxide include stimulation of cell division (proliferation) and cell maturation, stimulation of cell differentiation and formation of cell receptors, neovascularisation, formation of fibroblasts in the wound and thereby enhancement of collagen formation, etc.
Topical delivery of nitric oxide can be a very useful feature in various therapeutic or cosmetic applications including wound healing, treatment of skin or nail infections, sexual dysfunction etc.
U.S. Pat. No. 6,103,275 discloses a method for therapeutically applying nitric oxide, the method comprising bringing together a nitrite salt, a reductant and an acid with pKa between about 1 and about 4 at a body site.
The pH range at which the method should be used is not specified. However, the fact that the buffer components are referred to as acids may indicate that these compounds are predominantly present in the protonated form, therefore the pH of the composition should be substantially lower than 4. The presence of acids with pKa less than 4 (e.g. between 1 to 4) ensures good buffering capacity of the formulation at the required pH. Whilst incorporation of such acids is a convenient way of ensuring that pH is maintained at a level such that a continuous efficiency of converting nitrite to nitric oxide is maintained, there are disadvantages of introducing these acids into the system. Prolonged exposure of skin to any topical application that is buffered strongly at pH less than 4 is potentially harmful and should be avoided.
Other nitric oxide releasing systems have been disclosed. For example, U.S. Pat. No. 6,709,681 discloses a method of treating microbial infection, the method comprising mixing acidifying agent with a source of nitrite. In principle, this method is very similar to that disclosed in U.S. Pat. No. 6,103,275, i.e. mixing a source of nitrite with acids of pKa between 1 to 4. Importantly, the absence of strong reducing agents in the formulation disclosed in U.S. Pat. No. 6,709,681 does not ensure sufficient reducing power in the formulation. Consequently, generation of nitric oxide will be accompanied by direct generation of nitrogen dioxide according to the following mechanism:NO2−+H+→HNO2 2HNO2→NO+NO2+H2O
Whilst nitric dioxide may exert good antimicrobial properties, it does not have vasodilating properties nor is it capable of activation of the cell proliferation. It is therefore generally desirable to stop the direct generation of nitrogen dioxide by incorporating the reducing agent.
U.S. 2003012816 discloses a biocompatible polymerisable macromer composition comprising a macromer having at least one nitric oxide carrying region or nitric oxide modulating compound wherein the nitric oxide or the nitric oxide modulating compound is released from the macromer and wherein the macromer further comprises one or more regions selected from the group consisting of a water soluble region, a cell adhesion ligand and a polymerisable region. The disclosed macromers include acrylolyl-PEG-Cys-NO macromer, acrylolyl-PEG-Lys5-NO macromer, PEG-DETA-NO macromer, PVA-NH2-NO macromer, PVA-Cys-NO macromer and PVA-NO-bFGF macromer.